Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily of ligand-activated transcription factors. Three subtypes of PPARs have been cloned from the mouse and human: i.e., PPARalpha, PPARgamma and PPARdelta. In humans PPARgamma and PPARalpha are differentially expressed in organs and tissues (Willson et al. J. Med. Chem. 43:527-50 (2000) Nuclear receptors like PPAR possess DNA binding domains (DBDs) that recognized specific DNA sequences (called response elements) located in the regulatory regions of their target genes Mangelsdorf et al Cell 83:835-839 (1995)). Activation of PPARs modulates the expression of genes containing the appropriate respective peroxisome proliferator response elements (PPRE) in its promoter region. PCT WO/25226.
PPARgamma consists of three forms, PPARgamma1 which is broadly expressed in most tissues, PPARgamma2, is more restricted to adipose (white fat and brown fat) tissue, and PPARgamma3. PPARgamma3 is confined to adipose tissue, macrophages and colonic epithelium in rodent and human tissues (Mangelsdorf, and Evans. Cell (1995) 83:841-850; Spiegelman. Diabetes (1998) 47:507-514; Willson et al., J. Med. Chem. (2000) 43:527-550). The distribution of the other PPARs also varies in different tissues. Throughout this writing PPAR refers to any of these isoforms, subtypes or combination thereof. PPARgamma is functionally involved in intermediary metabolism of cells and tissues that express this nuclear receptor.
PPARgamma and PPARalpha and PPARdelta are differentially expressed in different organs and tissues. Activation of PPARgamma and/or PPARalpha and/or PPARdelta modulates the expression of genes involved in: 1) glucose and lipid metabolism, 2) the regulation of cell growth, differentiation and regulation of the mitotic cycle, 3) the inflammatory response in cells of the immune system, 4) suppression of components of the immune system that become activated in pathological situations, and 5) regulation of apoptosis (programmed cell death) in a variety of cell types. Impairment in these processes lead to pathophysiolgical conditions involving metabolic (endocrine) dysfunction, proliferative diseases, inflammatory diseases and degenerative diseases. (Pershadsingh, Expert Opin. Investig. Drugs 8(11):1859-1872 (1999)).
The precise mechanism whereby ligand activation of PPARs lead to changes in gene expression is poorly understood. Full activation of PPARgamma and/or PPARalpha and/or PPARdelta requires its functional dimerization with the retinoid X receptor (RXR) to form PPARgamma/RXR or PPARalpha/RXR or PPARdelta/RXR. The endogenous ligand for RXR is 9-cis-retinoic acid. Nutrient retinoids and retinoic acid such as 13-all-trans-retinoic acid are converted to 9-cis-, 11-cis-, or 13-cis-retinoic acid by ubiquitous intracellular isomerases (Warrell Jr et al., New Engl. J. Med. 329(3):177-189 (1993)). The full spectrum of genes that can be regulated by PPARalpha or PPARgamma or PPARdelta or their respective heterodimers remain to be defined.
PPAR agonists have been shown to inhibit the expression of inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), IL-1, IL-2, IL-6 in cells of the immune system including, T lymphocytes, B lymphocytes, monocytes, monocyte/macrophages, and splenocytes. PPARgamma agonists tend to suppress inflammation mediated by Th1 lymphocytes (Marx et al., J. Immunol. 164:6503 (2000); Padilla et al., Ann N.Y. Acad. Sci. 905:97 (2000); Clark et al., J. Immunol. (2000) 164:1364; Yang et al., J. Biol. Chem. (2000) 275:4541). However, the anti-inflammatory effects of PPARgamma are controversial. It has been reported that PPARgamma activators are not useful for the treatment of acute inflammation in db/db diabetic mice (Thieringer et al., J. Immunol. (2000) 164:1046). Thiazolidinedione-treated db/db mice challenged with lipopolysaccharide, a potent pro-inflammatory agent, displayed no suppression of cytokine production. Rather, their blood levels of TNF-alpha and IL-6 were elevated beyond the levels observed in control mice.
PPAR agonists have also been shown to inhibit proliferation and promote differentiation of a variety of normal and neoplastic cell types. Spiegelman et al., PCT/US97/22879, published Jun. 18, 1998, disclose methods for inhibiting proliferation of PPARgamma-responsive hyperproliferative cells by using PPARgamma agonists; numerous PPARgamma agonists are disclosed by Spiegelman et al., as well as methods for diagnosing PPARgamma-responsive cells. The method relates to superior efficacy of PPAR activators or co-activators of various subtypes in: 1) promoting apoptosis of neoplastic cells, 2) systemic anti-inflammatory effect by suppressing Th1-mediated inflammatory cytokines and promoting Th1 to Th2 phenotypic transition resulting in of immunosuppression, leading to prevention, amelioration or reversal of degenerative diseases.
Examples of diseases susceptible to the immunosuppressive effects of activators of PPARgamma, or PPARalpha, or PPARdelta, or co-activators of any of these subtypes are: inflammatory skin diseases (e.g. psoriasis, atopic dermatitis, eczema, acne vulgaris, and other dermatitides), neurodegenerative diseases (e.g. multiple sclerosis, Alzheimer's disease, Parkinson's disease), cardiovascular diseases (e.g. atherosclerosis, venous and arterial occlusive diseases, restenosis after invasive procedures, cardiomyopathy, myocardial fibrosis, congestive heart failure), pulmonary disorders (asthma, chronic obstructive pulmonary disease), angiogenesis and neovascularization in neoplastic and other diseases. The immune system includes T lymphocytes, B lymphocytes, monocytes, macrophages, monocyte/macrophages, macrophage-like cells (e.g. astrocytes in the brain, retinal pigmented epithelial cells in the retina), cells of myeloid origin in any tissue, in particular the bone marrow (stem cells, pre-promyelocytes, promyelocytes, myelocytes, granulocytes, plasma cells, mast cells, basophils, polymorphonuclear cells, eosinophils).
In humans, PPARgamma and PPARalpha and PPARdelta are differentially expressed in organs and tissues (Willson et al., J. Med. Chem. (2000) 43 (4):527-50). This heterogeneous distribution is particularly evident in the complex structure of the eye (Braissant et al., Endocrinology (1996) 137:354-366; Pershadsingh et al., Proceedings of the Society for Neurosciences. Miami Beach, USA, 1999). It can be difficult to predict what cells and diseases are influenced by PPARgamma and/or PPARalpha activity and/or PPARdelta due to the varied tissue distribution of expression of the various PPAR subtypes and the varied amounts of their respective proteins in various cells (Escher et al., Endocrinology 142(10):4195-202 (2001); Braissant O, Wahli W, Endocrinology 139(6):2748-54 (1998)). Further, some PPAR subtypes are expressed in some cells while in a normal state, but not expressed or expressed to a lesser or greater degree by the abnormal cells, or visa versa. Specifically, PPARgamma and PPARalpha are differentially expressed in diseased versus normal cells. For example PPARgamma is expressed in normal human keratinocytes but not in normal human dermal fibroblasts (Ellis et al., Arch. Dermatol. (2000) 136:609-616). PPARgamma has been shown to be expressed in a greater amount in level was increased in the subcellular cytosolic fraction of Alzheimer's disease brains, compared to control brains (Kitamura et al., Biochem. Biophys. Res. Commun. 1999; 254:582-586).
The activity of PPARgamma or PPARalpha or PPARdelta depends on the degree to which the receptor protein is phosphorylated and/or on the conformation of the receptor. It has been proposed that phosphorylation could alter interactions with protein cofactors of PPARgamma which act as corepressors or coactivators. Nuclear receptors function as “ligand-gated” platforms for the assembly of these cofactors into large protein complexes on specific DNA sequences (Spiegelman Diabetes (1998) 47:507-514). Some of these coactivator proteins (CBP/p300, SRC1, pCAF) have histone acetyltransferase activity that functions to “open” the configuration of chromatin, allowing more efficient transcription. Others act as deacetylases which oppose the effects of acetyltransferases. Similar arguments apply to PPARalpha modulation of gene transcription. One theoretical problem is whether the nuclear receptor coactivators or corepressors identified to date are selective for particular PPAR receptors, and this remains unknown (Spiegehnan B M. Diabetes (1998) 47:507-514). In fact, these coactivators or corepressors have multiple modes of action and hence, it is not clear which cofactors are more important for the function of any particular receptor (Puigserver et al., Science (1999) 286:1368-1371. It is also not obvious how the tremendous specificity of biological actions of the individual nuclear receptors are generated (Spiegelman, Diabetes (1998) 47:507-514). Consequently, the full spectrum of nuclear cofactors that regulate the transcriptional activity of PPARgamma and/or PPARalpha or PPARgamma/RXR and/or PPARalpha/RXR remains to be defined. The way in which a tissue expressing PPARgamma and/or PPARalpha and/or PPARdelta may respond to a particular ligand, and a pathological state will be attenuated, arrested, accentuated or worsened by said ligand can vary for example in the case in which a single ligand activates both PPARgamma and PPARalpha to similar degrees, i.e. a co-activator of both PPARgamma and PPARalpha (or similarly, both PPARgamma and PPARdelta or PPARalpha and PPARdelta).
Until recently, the genes regulated by PPARs were those believed to be predominantly associated with lipid and glucose metabolism. Recently, an immunomodulatory role for PPARgamma and PPARalpha has been described (Shu et al., Biochem. Biophys. Res. Commun. (2000) 267(1):345-9). The immunomodulatory/immunological mechanisms underlying inflammatory diseases mediated by or related to T lymphocyte activation are not well understood. Immunosuppressive agents capable of blocking various steps of the immune response have been utilized to prevent, ameliorate or reverse the inflammatory process, often by downregulating critical nuclear transcription factors that, in turn, regulate the expression of genes encoding inflammatory cytokines. Production of inflammatory cytokines occur in hypertriglyceridemic and other dyslipidemic states, e.g. diabetes mellitus.
Both PPARalpha and PPARgamma activators have been shown, independently, to suppress expression of these inflammatory regulators, inhibit proliferation and promote apoptosis of pathological cellular phenotypes. Paradoxically and unexpectedly, the opposite case occurs wherein the therapeutic compositions are administered in the treatment of degenerative disease such as multiple sclerosis (a neurodegenerative) or retinopathies and retinitis (retinal degenerative diseases), in which prevention of apoptosis is the operative mechanism. Therefore, in these disease states, activation of PPARalpha and PPARgamma by suppressing transcription of inflammatory cytokines, prevents apoptosis of the target cell and promotes survival of the non-pathological cellular phenotype. For example, in the case of multiple sclerosis, an autoimmune T lymphocyte-mediated disease, the target cell sustaining the pathological insult is the myelin sheath (oligodendrocyte) in the central nervous system. The pathological cellular phenotypes are amnestic T lymphocytes lacking immune recognition of oligodendrocytes, and inappropriately activated microglia, resulting in inappropriately activation and production of harmful inflammatory cytokines (Zhang et al., Mult. Scler (2000) 6:3-13). PPARgamma activation can inhibit neuronal apoptosis and promote neuronal protection through the upregulation of neuronal apoptosis inhibitory protein (Magun et al., Diabetes (1998) 47:1948-52). Indeed, PPARgamma activation protects cerebellar granule cells from cytokine-induced apoptotic cell death (Heneka et al., J. Neuroimmunol. (1999) 100:156-68). Moreover, PPARalpha has been shown to suppress inflammatory cytokines and nuclear factors in monocyte/macrophages. A similar mechanism involving suppression of inflammatory cytokine production by microglia would prevent oligodendrocyte apoptosis. Finally, combined PPARalpha/PPARgamma activation could promote Th1/Th2 differentiation as a final common pathway to inhibit apoptosis of the non-pathological phenotype and promotion of neuronal protection (Giorgini et al., Horm. Metab. Res. (1999) 31:1-4; Clark et al., J. Immunol. (2000) 164:1364-71).
PPARgamma interactions with co-activators and co-repressors tend to be ligand-specific. For example, the natural PPARgamma ligand, 15-deoxy-delta-12,14-prostaglandin J2 can induce the receptor-ligand complex to interact with the cofactors: SRC-1, TIF2, AMB-1, p300, TRAP220/DRIP205, whereas, under the same conditions the anti-diabetic thiazolidinedione, troglitazone, a synthetic PPARgamma ligand does not. Therefore, ligand binding may alter PPARgamma structure in a ligand-type specific way, resulting in distinct PPARgamma-coactivator interactions (Kodera et al., J. Biol. Chem. (2000) 275(43):33201-33204. By analogy, a similar mechanism would provide ligand-specific control of gene expression in the case of PPARalpha activation or PPARdelta activation.